This application relates to agents capable of specifically recognizing GPlba, a key receptor required for platelet adhesion and aggregation. More specifically, this application relates to monoclonal antibodies and derivatives thereof capable of specifically recognizing both the murine and the human GPlba. These monoclonal antibodies and derivatives are particularly useful in the treatment or prevention of thrombosis as well as research tools.
This application refers to and comprises nine biological deposits (hybridomas) accepted at the International Depositary Authority of Canada on October 7, 2008 under Accession Numbers 071008-01 (NIT A1 clone), 071008-02 (NIT B1 clone), 071008-03 (NIT C1 clone), 071008-04 (NIT D1 clone), 071008-05 (NIT E1 clone), 071008-06 (NIT F1 clone), 071008-07 (NIT G1 clone), 071008-08 (NIT H1 clone), 071008-09 (NIT I1 clone). The viability of these clones was tested on October 15, 2008. These biological deposits have been made in accordance with the Budapest Treaty. The content of these biological deposits are incorporated herein by reference.
The present invention relates to monoclonal antibodies, and more specifically, to monoclonal antibodies that specifically recognizing platelet GPlbα. These antibodies are useful, for instance, in antithrombotic therapy, in diagnostics as well as in animal models of thrombosis, hemostasis, thrombocytopenia, and anti-angiogenesis.
Platelet adhesion and aggregation are key events required to arrest bleeding. However, the same processes also contribute to the generation of thrombi within atherosclerotic arteries such as the coronary or cerebral arteries, which is the worldwide leading cause of morbidity and mortality.
Platelet GPlbα is an important platelet receptor which mediates platelet adhesion to the vessel wall by interacting with, for example, the von Willebrand factor (Wolfgang et al., 2006, PNAS, 103:16900-16905). Once a platelet is anchored to the vessel wall, it facilitates the aggregation of other platelets (
Antagonists targeting GPlbα would provide significant advantages as compared with other anti-platelet drugs such as those antagonists to β3 integrin (also known as GPIIbIIIa), such as ReoPro™ approved 1994, Integrinlin™ approved 1998, Aggrastat™ approved 1998, already in the market. The prevailing view is that antagonists of GPIbα may prevent the thrombus formation at an earlier stage than β3 integrin antagonists, and as such would be more potent and would exhibit less side effects than β3 antagonists (e.g. bleed tendency observed in antagonists to the GPllbllla receptor).
Monoclonal antibodies against GPlbα are readily available and currently used in the art. However, these antibodies have been made in wild-type animals (e.g. mice) and, as such, do not recognize the mouse GPlbα. Since mouse and human GPlbα are similar polypeptides (e.g. they possess an important degree of homology), the use of a wild-type mouse to generate the monoclonal antibodies limits the repertoire of the antibodies produced to epitopes present on the human GPlbα and absent on the mouse GPlbα.
In light of the above, it would be highly desirable to be provided with antibodies or compositions derived therefrom capable of specifically recognizing the platelet GPlbα receptor. Those agents would preferably specifically recognized both the human and the mouse GPlbα receptors on platelets. Those agents are preferably antibodies, such as monoclonal antibodies, capable of specifically recognizing and/or neutralizing the platelet GPlbα receptor.
According to one aspect, the present invention relates to an hybridoma cell line having IDAC Accession No. 071008-01 (NIT A1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-02 (NIT B1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-03 (NIT C1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-04 (NIT D1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-05 (NIT E1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-06 (NIT F1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-07 (NIT G1 clone) filed on October 7, 2008, an hybridoma cell line having IDAC Accession No. 071008-08 (NIT H1 clone) filed on October 7, 2008 and/or an hybridoma cell line having IDAC Accession No. 071008-09 (NIT I1 clone) filed on October 7, 2008.
According to a further aspect, the present invention also relates to an isolated and purified antibody produced by the hybridoma cell line described herein. In an embodiment, the isolated and purified antibody is humanized. In another embodiment, the isolated and purified antibody is labeled with a detectable marker or a conjugate. In another embodiment, the detectable marker is at least one of a gold marker, a fluorescent marker and radioactive marker. In a further embodiment, the conjugate is at least one of biotin, peroxidase, alkaline phosphatase, glucose oxidase and phycobiliprotein. According to yet a further embodiment, the isolated and purified antibody is recognized by a labeled antibody.
According to another aspect, the present invention relates to a fragment of an isolated and purified antibody produced by the hybridoma cell line described herein. In an embodiment, the fragment is humanized. In still another embodiment, the fragment is labeled with a detectable marker or a conjugate. In yet a further embodiment, the detectable marker is at least one of a gold marker, a fluorescent marker and a radioactive marker. In still another embodiment, the conjugate is at least one of biotin, peroxidase, alkaline phosphatase, glucose oxidase and phycobiliprotein. In yet a further embodiment, the fragment is recognized by a labeled antibody.
According to still another aspect, the present invention provides a pharmaceutical composition comprising the isolated and purified antibody of any described herein or the fragment described herein and a pharmaceutical acceptable carrier. In an embodiment, the pharmaceutical composition is for inhibiting platelet aggregation. In another embodiment, the pharmaceutical composition is for the treatment of thrombosis. According to another aspect, the present invention provides the use of the isolated and purified antibody of any described herein or the fragment described herein as a medicament, or as a diagnostic tool of a thrombus.
According to yet another aspect, the present invention provides a solid phase having attached thereto the isolated and purified antibody described herein or the fragment described herein. In an embodiment, the solid phase is used for the separation or isolation of a GPIbα receptor. In another embodiment, the solid phase is used for the separation or isolation of a platelet. In still another embodiment, the solid phase comprises at least one of a gel, a hydrogel, a resin, a bead, nitrocellulose, a nylon membrane, a micrometer plate, a culture flask, a polymeric material and glass.
According to another aspect, the present invention provides a method for preventing the formation of a thrombus and/or the aggregation of platelets in an individual, said method comprising administering the isolated and purified antibody described herein or the fragment described herein in the individual, thereby preventing the formation of the thrombus and/or the aggregation of platelets. In an embodiment, the administration of the isolated and purified antibody or the fragment limits the size of the thrombus and/or the aggregation of platelets. In still another embodiment, the administration of the isolated and purified antibody or the fragment limits the number of thrombi in the individual. In yet another embodiment, the individual is also administered with a further antithrombotic agent.
According to still another aspect, the present invention provides use of the isolated and purified antibody described herein or the fragment described herein for the prevention of the formation of a thrombus and/or the aggregation of platelets in an individual.
According to still yet another aspect, the present invention provides a method of resolving thrombosis in an individual having a thrombus, said method comprising administering the isolated and purified antibody described herein or the fragment described herein in the individual, thereby resolving thrombosis in the individual. In an embodiment, the administration of the isolated and purified antibody or the fragment limits the size of the thrombus. In another embodiment, the isolated and purified antibody or the fragment limits the number of thrombi. In yet another embodiment, the individual is also administered with a further antithrombotic agent.
According to another aspect, the present invention provides use of the isolated and purified antibody described herein or the fragment described herein for the resolution of thrombosis in an individual having a thrombus.
According to another aspect, the present invention also provides a method for determining the presence of a thrombus in an individual, said method comprising administering the isolated and purified antibody described herein or the fragment described herein to the individual, allowing the isolated and purified antibody or the fragment to form a complex with a GPIbα receptor on a platelet and determining the presence of the complex. In an embodiment, the individual has received an antithrombotic agent prior to or after the administration of the isolated and purified antibody or the fragment. In another embodiment, the determination of the presence of the complex is repeated in time. In another embodiment, the individual is an animal. In a further embodiment, the animal is a rodent and, in a yet a further embodiment, the rodent is a mouse.
According to still another aspect, the present invention provides use of the isolated and purified antibody described herein or the fragment described herein for a method for determining in vivo the presence of a thrombus in an individual.
According to another aspect, the present invention provides a method for determining platelet count in a sample from an individual, said method comprising contacting the isolated and purified antibody described herein or the fragment described herein with the sample from the individual to form a complex between (i) the isolated and purified antibody of the fragment and (ii) the platelet and detecting the presence of the complex to determine the platelet count in the sample.
According to another aspect, the present invention provides a method for tracking a platelet in an individual, said method comprising administering the isolated and purified antibody described herein or the fragment described herein to the individual, allowing for a complex between (i) the isolated and purified antibody or the fragment and (ii) the platelet to be formed, detecting the location of the complex to track the platelet. In an embodiment, the detection is repeated in time. In another embodiment, the individual is an animal. In a further embodiment, the animal is a rodent and, in a yet a further embodiment, the rodent is a mouse.
According to yet another aspect, the present invention provides use of the isolated and purified antibody described herein or the fragment described herein for a method for tracking in vivo a platelet in an individual.
This application relates to agents capable of specifically recognizing GPlbα, a key receptor required for platelet adhesion and aggregation. As used herein, the terms "GPIbα" and "GP1ba" are used interchangeably and refer to the same receptor that is present on platelets. The GPIbα GenBank reference number for the human version is NP_000164 and for the mouse version is NP_034456. More specifically, this application relates to monoclonal antibodies (mAbs) and derivatives thereof capable of specifically recognizing both the murine and the human GPIbα. In an embodiment, these antibodies can also recognize the pig, rabbit, rat, goat, and sheep GPlbα. These monoclonal antibodies and derivatives are particularly useful in the treatment and/or prevention of thrombosis as well as research tools. These mAbs can also be useful in the diagnosis for several GPlbα-related diseases such as Bernard-Soulier syndrome and immune thrombocytopenia.
GPlbα interaction with VWF play a critical role for platelet adhesion and aggregation. Since GPlbα is involved in the early stages of thrombosis, antagonists of platelet GPlbα have great potential for anti-thrombotic therapy and prevent vessel occlusion, which would provide significant advantages as compared with other anti-platelet drugs such as those antagonists to β3 integrin. However, in the art, there is no GPlbα antagonist commercially available. Current monoclonal antibodies (mAbs) against human GPlbα have been made in wild-type mice. However, since mouse and human GPlbα are very similar polypeptides and since the immune system usually does not response to self antigen (such as GPIbα), this approach limits the repertoire of the antibodies produced to epitopes presented on the human GPlbα and absent on the mouse GPlbα. Monoclonal antibodies produced using conventional techniques therefore cannot recognize mouse GPlbα. Using GPIbα -gene deficient mice immunized with wild-type platelets, Applicant firstly generated anti-GPlbα mAbs which recognize not only mouse but also human GPIbα. The mAbs produced inhibit platelet adhesion and thrombus growth. Compare with current available antibodies, these monoclonal antibodies are believed to recognize different epitopes of GPIbα on platelets. These antibodies can be potential anti-thrombotic drugs and useful reagents for hemostasis and thrombosis research.
Traditional methods to generate monoclonal antibodies (mAbs) against human proteins usually use wild-type BALB/c mice. The antigenicity of a human protein, and thus the ability to generate mAbs using such methods, will be based on the difference between the human and murine protein sequences. Since GPIbα is relatively highly conserved between these two species (∼50-60% identity), the repertoire of antibodies against human GPlbα integrin in wild-type BALB/c mice is limited. In the art, the mAbs generated by the traditional method do not recognize both murine and human GPIbα.
In order to circumvent this limitation, Applicant has generated novel mAbs by using GPlbα-deficient BALB/c mice, which do not express GPlbα and can thus produce antibodies against any portion of GPlbα. The repertoire of antibodies against GPlbα using this methodology is much larger compared to using wild-type mice since an antibodies can be generated against the entire GPlbα protein, even in the regions highly conserved between the murine and the human version. The GPlbα knocked-out mice have then been transfused with wild-type platelet (e.g. wild-type mouse platelets). Hybridomas have been generated with the spleens of these mice. Using this technology, nine monoclonal antibodies (mAbs) (NIT-A, B, C, D, E, F, G, H and I) from GPlbα deficient mice after wild-type mouse platelet transfusions. These antibodies specifically bind to mouse GPlbα. Three clones (NIT-A, B and F) also bind human and rat platelets. Two clones (NIT-C and D) also bind pig platelets. As it will be shown herein, some clones can completely inhibit human platelet aggregation induced by ristocetin, therefore may have potential for antithrombotic therapy (e.g. inhibitory mAbs). The other antibodies produce do not affect platelet adhesion and aggregation (neutral mAbs). The mAbs produced (including both inhibitory and neutral mAbs) have potential to be used as diagnostic reagents (e.g. Bernard-Soulier syndrome and immune thrombocytopenia). The radioactive isotope labeled neutral mAbs can be used to track platelet circulation in vivo. These mAbs are important research tools for thrombosis and hemostasis including animal models of these human diseases.
Accordingly, the present application relates to the hybridoma cell lines that have been produced according to the method described herein. Some of these hybridoma cell lines have been deposited International Depositary Authority of Canada on October 7, 2008 under Accession Numbers 071008-01 (NIT A1 clone), 071008-02 (NIT B1 clone), 071008-03 (NIT C1 clone), 071008-04 (NIT D1 clone), 071008-05 (NIT E1 clone), 071008-06 (NIT F1 clone), 071008-07 (NIT G1 clone), 071008-08 (NIT H1 clone), 071008-09 (NIT I1 clone).
The present application also relates to the antibodies produced by the hybridoma cell lines described herewith as well as derivatives and fragments of those antibodies. Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. Within the variable region of the light chain is a C-terminal portion known as the J region. Within the variable region of the heavy chain, there is a D region in addition to the J region. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding. Proceeding from the amino-terminus, these regions are designated CDR1, CDR2 and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined by Kabat et al. (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)).
Antibody derivatives include, but are not limited to, humanized antibodies. As used herein, the term "humanized antibody" refers to an immunoglobulin that comprises both a region derived from a human antibody or immunoglobulin and a region derived from a non-human antibody or immunoglobulin. The action of humanizing an antibody consists in substituting a portion of a non-human antibody with a corresponding portion of a human antibody. For example, a humanized antibody as used herein could comprise a non-human region variable region (such as a region derived from a murine antibody) capable of specifically recognizing GPlbα and a human constant region derived from a human antibody. In another example, the humanized immunoglobulin can comprise a heavy chain and a light chain, wherein the light chain comprises a complementarity determining region derived from an antibody of non-human origin which binds GPlbα and a framework region derived from a light chain of human origin, and the heavy chain comprises a complementarity determining region derived from an antibody of non-human origin which binds GPlbα and a framework region derived from a heavy chain of human origin.
As used herein, the present application also relates to fragments of the monoclonal antibodies described herein. As used herein, a "fragment" of an antibody (e.g. a monoclonal antibody) is a portion of an antibody that is capable of specifically recognizing the same epitope as the full version of the antibody. In the present patent application, antibody fragments are capable of specifically recognizing GPlbα. Antibody fragments include, but are not limited to, the antibody light chain, single chain antibodies, Fv, Fab, Fab' and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab')2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding the heavy chain of an F(ab')2 fragment can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. Antibody fragments can also be humanized. For example, a humanized light chain comprising a light chain CDR (i.e. one or more CDRs) of non-human origin and a human light chain framework region. In another example, a humanized immunoglobulin heavy chain can comprise a heavy chain CDR (i.e., one or more CDRs) of non-human origin and a human heavy chain framework region. The CDRs can be derived from a non-human immunoglobulin.
In research, the capacity of antibodies to specifically recognize an epitope on a specific polypeptide is used to detect (e.g. visualize) the polypeptide. The antibodies described herein can be used for the detection of GPlbα in immunohistology, immunofluorescence, immunoblot, electron microscopy, and/or immunoprecipitation. In order to do so, the antibodies must first be labeled or adapeted to be used with a second (or third) antibody that is labeled. In an embodiment, the antibodies and fragments presented herein can be labeled with a detectable marker or a conjugate. This is particularly advantageous when the antibodies are to be used as diagnostic or research tools. The detectable markers include, but are not limited to gold (for electron microscopy applications), a fluorescent marker (for immunofluorescence and flow cytometry applications) and a radioactive marker (for diagnostic purposes). For example, if the antibodies, derivatives or fragments are labeled to a radioisotope, they could also be used in in vino diagnostic methods. Conjugates used as labels are particularly useful in assays involving enzymes (such as ELISA). Useful conjugates are numerous and include biotin, peroxidase, alkaline phosphatase, glucose oxidase and phycobiliprotein, and radioisotope. The antibodies and fragments thereof could also be detected indirectly by coupling a label to another antibody that specifically recognized the anti-GPlbα antibodies or the fragments thereof. In this particular embodiment, the use of a further antibody can increase the signal obtained.
As antibodies (as well as derivatives or fragments thereof) are extensively used in the research setting for purifying or isolating polypeptides, the antibodies described herein (as well as derivatives or fragments thereof) could also be successfully used for the purification (e.g. immunopurification or immunoprecipitation) or the isolation of the GPlbα (human or mouse) polypeptide and/or the platelet expressing the GPlbα. Antibodies are also used in assay for detecting the presence of a particular polypeptide in a sample, such as a ELISA, a sandwich ELISA or a polypeptide microarray. As such, the antibodies described herein could also be used for the detection of GPlbα (human and/or mouse) in a sample. For both of these applications (purification and/or detection), the antibodies are usually linked to a solid carrier. Accordingly, in one additional embodiment, the present application provides a solid phase having attached thereto the antibodies described herein (as well as derivatives thereof and fragments thereof). The solid phase can be, but is not limited to a gel, a hydrogel, a resin, a bead, nictrocellulose, nylon membrane, amicrometer plate, a culture flask, a polymeric material and/or glass.
In a further embodiment, the present application also contemplates the use of the antibodies of fragments thereof described herein in pharmaceutical compositions. The pharmaceutical compositions optionally also comprises a pharmaceutical acceptable carrier. As used herein a "carrier" or "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more antibody or fragment thereof to an individual. Because antibodies or fragments thereof are large compounds, there are preferably injected in the individual. A pharmaceutical carrier is generally selected to provide for the desired bulk, consistency, etc., when combined with components of a given pharmaceutical composition, in view of the intended administration mode. Typical pharmaceutical carriers include, but are not limited to binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
In yet a further embodiment, the present application also contemplates the use of the antibodies of fragments thereof described herein in diagnostic compositions. The diagnostic compositions optionally also comprises an acceptable carrier that is suited for the diagnostic use of the antibodies.
Antibodies or fragments thereof disclosed in the present application may be administered with a pharmaceutically-acceptable carrier in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to individuals. Although injection is preferred, any appropriate route of administration may be employed, for example, oral, intravenous, perenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, or aerosol administration. Therapeutic formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found in, for example,
In addition, the term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (dose) effective in treating an individual. It is also to be understood herein that a "pharmaceutically effective amount" may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.
A therapeutically effective amount or dosage of an antibody or fragment thereof disclosed herein or a pharmaceutical composition comprising the antibody or fragment thereof, may range from about 0.001 to 30 mg/kg body weight, with other ranges including about 0.01 to 25 mg/kg body weight, about 0.025 to 10 mg/kg body weight, about 0.3 to 20 mg/kg body weight, about 0.1 to 20 mg/kg body weight, about 1 to 10 mg/kg body weight, 2 to 9 mg/kg body weight, 3 to 8 mg/kg body weight, 4 to 7 mg/kg body weight, 5 to 6 mg/kg body weight, and 20 to 50 mg/kg body weight. In other embodiments, a therapeutically effective amount or dosage may range from about 0.001 to 50 mg total, with other ranges of the invention including about 0.01 to 10 mg, about 0.3 to 3 mg, about 3 to10 mg, about 6 mg, about 9 mg, about 10 to 20 mg, about 20-30 mg, about 30 to 40mg, and about 40 to 50 mg.
Because of the intrinsic properties of the antibodies disclosed herein, the pharmaceutical compositions are particularly useful for the inhibition of platelet adhesion and aggregation (such as a thrombus or a blood clot) in an individual and/or the treatment or prevention of thrombosis in an individual.
According to an embodiment, there is provided a method for the prevention of the formation of a blood clot in an individual. In this particular embodiment, only the antibodies that are capable of limiting the interaction of GPlbα (human or mouse) with its cognate ligand (such as the von Willebrand factor) can be successfully used. The method comprises the administration of the antibody described herein as well as derivatives thereof and/or fragments thereof or the administration a pharmaceutical composition as described herein. In a further embodiment, the administration of the antibody or the pharmaceutical composition does not completely prevent the formation of the blood clot or thrombus but limits its size. In a further embodiment, the administration of the antibody or the pharmaceutical composition does not completely prevent the formation of all the blood clots or thrombi in an individual by limits their number. In yet a further embodiment, the individual could also be administered with another antithrombotic agent, such as those commonly used in the art (for example heparin or an antagonist of a β3 integrin such as ReoPro™). Use of the antibodies described herein or the pharmaceutical compositions described herein for the prevention of the formation of a blood clot or thrombus is also contemplated.
According to an embodiment, there is provided a method for the resolution of thrombosis in an individual having at least one blood clot. As used herein, the term "resolution" refers to the subsidence of thrombosis. In this particular embodiment, preferably the antibodies that are capable of limiting the interaction of GPlbα (human or mouse) with its cognate ligand (such as the von Willebrand factor) can be successfully used. The method comprises the administration of the antibody described herein as well as derivatives thereof and/or fragments thereof or the administration a pharmaceutical composition as described herein. In a further embodiment, the administration of the antibody or the pharmaceutical composition does not completely eliminate the blood clot or thrombus by limits its size. In a further embodiment, the administration of the antibody or the pharmaceutical composition does not completely eliminate all the blood clots or thrombi in an individual by limits their number. In yet a further embodiment, the individual could also be administered with another antithrombotic agent, such as those commonly used in the art. Use of the antibodies described herein or the pharmaceutical compositions described herein for the resolution of thrombosis in an individual is also contemplated.
According to an embodiment, there is provided a method for determining the presence of a platelet rich thrombi (blood clot) in an individual. Platelet rich thrombi or white thrombi are the major cause of vessel occlusion in arteries and are the most common mechanism leading to myocardial or cerebral infarction (i.e. heart attack and stroke). In this particular embodiment, antibodies that are capable of recognizing platelet specific proteins (e.g. GPIbα (human or mouse) which is abundant on platelet surface) can be successfully used. The method comprises the administration of the antibody described herein as well as derivatives thereof and/or fragments thereof. The method also comprises allowing the antibody to form a complex. The method further comprises detecting the complex. The complex can be directly detected by labeling the antibody with an appropriate marker. The complex can also be detected indirectly by, for example, the administration of a second (or third) labeled antibody capable of recognizing the first antibody. The determination of the presence of the complex can be done once, or it can be repeated in time to determine the evolution or regression of the clot or thrombus. In an embodiment, the individual has developed a platelet rich thrombus (blood clot) prior to the administration of the antibody. In yet a further embodiment, the individual could also be administered with an antithrombotic agent, such as those commonly used in the art. In this particular embodiment, the detection, especially if it is repeated in time (e.g. prior to and after the administration of the antithrombotic agent), will generate valuable information about the efficiency of the antithrombotic drug to resolve the thrombosis. In an embodiment, the individual is an animal. In a further embodiment, the animal is a rodent, such as a mouse or a rat. Use of the antibodies described herein for the detection of a clot or thrombus in an individual is also contemplated.
According to an embodiment, there is provided a method for determining platelet count in sample from an individual. In this particular embodiment, antibodies that are capable of specifically recognizing GPIbα (human or mouse) can be successfully used. The method comprises contacting the antibody described herein as well as derivatives thereof and/or fragments thereof with the sample. The method also comprises allowing a complex between the antibody and the platelet to be formed and detecting such complex. The complex can be directly detected by labeling the antibody with an appropriate marker. The complex can also be detected indirectly by, for example, the administration of a second (or third) labeled antibody capable of recognizing the first antibody.
According to an embodiment, there is provided a method for tracking a platelet in an individual. In this particular embodiment, antibodies that are capable of specifically recognizing GPlbα (human or mouse) can be successfully used. The method comprises administering the antibody described herein as well as derivatives thereof and/or fragments thereof with the sample. The method also comprises allowing a complex between the antibody and the platelet to be formed and detecting such complex. The complex can be directly detected by labeling the antibody with an appropriate marker. The complex can also be detected indirectly by, for example, the administration of a second (or third) labeled antibody capable of recognizing the first antibody. The tracking of the platelet can be done once, or it can be repeated in time to determine the behavior of the platelet. In an embodiment, the individual has developed a blood clot prior to the administration of the antibody. In yet a further embodiment, the individual could also be administered with an antithrombotic agent, such as those commonly used in the art. In this particular embodiment, the detection, especially if it is repeated in time (e.g. prior to and after the administration of the antithrombotic agent), will generate valuable information about the effects of the antithrombotic drug to on the behavior of platelets. In an embodiment, the individual is an animal. In a further embodiment, the animal is a rodent, such as a mouse or a rat. Use of the antibodies described herein for the tracking of a platelet in an individual is also contemplated.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
Monoclonal antibodies were produced in accordance with the scheme illustrated in
Monoclonal antibodies were produced and purified according to standard methods. Briefly, antibody positive hybridoma cells were transfered into commercial HyQ ADCF-MAb sera-free medium (HyClone, Logan, UT, USA) in cell culture flasks (500 ml). Hybridoma cells were allowed to grow until they die, then the medium was collected. The medium was centrifuged at 5000 rpm, for 20 minutes at room temperature to remove the cells. The supernatant was collected, and diluted 1:1 with commercial ImmunoPure™ IgG Binding Buffer (Pierce Biotechnology, Inc. Rockford, IL, USA), then passed through a Protein G coupled sepharose column. Subsequently, the column was washed with binding buffer, and antibody was eluted after adding commercial ImmunoPure™ IgG Elution Buffer (Pierce Biotechnology, Inc.). The fractions of antibodies in elution buffer were collected in individual Eppendorf™ tubes. The optical density (OD) of each fraction was measured at A280 (to detect proteins), and the high concentration fractions were dialysed using a dialysis membrane in phosphate buffered saline.
Washed mouse platelets (1x 106) were incubated one hour with PBS (control), or a mAb produced according to the protocol set forth in Example I. The platelets were then stained with FITC-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG2b or IgG3 (Sigma) for 45 min. Samples were analyzed by flow cytometry (FACScan™ Becton Dickinson).
As indicated on
As shown in Example II, the monoclonal antibodies produced in Example I were shown to be capable of recognizing mouse platelets.
In order to assess if the monoclonal antibodies produced in Example I are also capable of recognizing human platelets, washed human platelets (1x 106) were incubated one hour with PBS (control), or a mAb produced according to the protocol set forth in Example I. The platelets were then incubated with FITC-labeled goat anti-mouse anti-GPlbα antibodies for 45 min. Samples were analyzed by flow cytometry. As shown on
In order to assess if the monoclonal antibodies produced in Example I are also capable of cross-reacting with rat platelets, washed rat platelets (1x 106) were incubated with PBS (control), or a mAB produced according to the protocol set forth in Example I. The platelets were then incubated with FITC-labeled goat anti-mouse IgG for 45 min and analyzed by flow cytometry. As shown in
In order to assess if the monoclonal antibodies produced in Example I are also capable of cross-reacting with pig platelets, washed pig platelets (1x 106) were incubated with PBS (control), or a mAB produced according to the protocol set forth in Example I. The platelets were then incubated with FITC-labeled goat anti-mouse IgG for 45 min and analyzed by flow cytometry. As shown in
Mouse platelets (
Human platelets (
Fluorescently labeled heparin (40 IU/mL) anti-coagulated human whole blood was incubated with PBS (control;
Fluorescently labeled heparin (40 IU/mL) anti-coagulated mouse whole blood was incubated with PBS (control;
Platelets were fluorescently labeled and infused into 3- to 5-week-old anesthetized C57BL/6 WT mice. The mesentery was exposed through a midline abdominal incision. Vessel injury was generated by using a filter paper soaked with a FeCl3 solution and placed over the vessel. The paper is then removed, and the vessel is covered with saline. Vessels were monitored for several minutes to 40 minutes after FeCl3 treatment or until occlusion. To determine platelet tethering to injured vessels, FeCl3 is superfused over mesenteric arterioles, and the number of fluorescently labeled platelets tethering to vessel walls is counted over a stretch of vessel as previously described (
As shown on
BALB/c mice platelets are enumerated on day 1 (basal count, 100%). Thrombocytopenia is induced by intravenously injection of one of three doses of anti-monoclonal antibodies 5 µg, 10 µg, 15 µg per mouse (n = 6) of the antibodies produced by the protocol of Example I. Platelets are again enumerated 24 hours after antibody injection (day 2). Thrombocytopenia induction is represented in as the percent decrease in platelet count 24 hours after antibody injection. This method to induce thrombocytopenia has been already described in
Monoclonal antibodies capable of inducing thrombocytopenia in mice are considered as potential anti-thrombotic agents and research reagents for immune thrombocytopenia.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
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